I am having problem with real time (SYBR green) amplification of gene. I have designed primers spanning exon-intron junction. Primer con, was 1picomole. I have used 5micrograms of RNA for cDNA conversion, diluted this cDNA ten times for reaction. Here sharing the amplification plot. Please suggest where things are going wrong. Thanks in advance..
This is not an amplification plot. I will suggest running the products after QPCR on agarose gel to see if you get expected product size.
Agree with the above. That is the melting curve, a derivative of which is useful to decide if you are getting one or multiple products - as is running the products out on a gel. Show the amplification curve, or the derivative of the melt curve if you want further comments.
The melting curve do not provide useful information in order to answer your question. I suggest you upload an amplification curve. But, the analysis of your melting curve clearly show that a problem is occuring.
Hi,
See your amplification plot and run the PCR product on the gel. What about melt curves of other genes with the same template. One more thing is dont use more concentrated RNA for your cDNA synthesis.
As mentioned above, you just present melting curve. But it seems anyway that there's no amplification. I suggest you to run your PCR in the same condition with a positive control (if available, cloned target for exple), negative control, different cDNA concentrations and another set of primers (which is known to work properly) targeting another gene, (housekeeping gene for instance, or with a high expression's level), in order to check your cDNA integrity. (and don't forget to check your PCR efficiency)
I agree with the others, you don't have any amplification here. I would also recommend using less RNA for cDNA reaction (150 ng to 1 µg). Check the cDNA with primers you know are working. Then try different annealing temperatures and MgCl2 concentrations for your primer pair in a normal PCR cycler (gradient, if you have) and check on an agarose gel for your product. If that looks good, go back to the real-time machine.
Thank you everybody for your suggestions. I have checked the efficiency of primers by semi q pcr and iam getting product at same tm. Here sharing the melt curve. Please help to spot the problem.
I suggest you start troubleshooting by running a house keeping gene to check that your reagents work well by semi quantitative PCR then real-time PCR. I also suggest re-measuring your DNA and RNA concentrations to check if any degradation has happened.
recently i again run real time PCR. Iam getting the pattern as shown in 1st pic. in some samples abut proper amplification pattern in treated samples. I have used equal cone of RNA for both and verified tm by semi q pcr.
Please show amplification curves.
If the concentration of your target cDNA is v low in the untreated samples, the primers may be binding non-specifically. What kind of Ct do you get for both?
target cDNA cone is same in both as looked from GAPDH amplification pattern. Here sharing the melt curve image of control and treated sample. Primers in control samples were verified by semi q pcr and there was amplification.
Did I get that right, that you don't see amplification from the control sample in the real-time machine but in a regular PCR machine? How many cycles did you run and what are the Ct values for the treated sample, which obviously is amlified in the real-time machine?
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